WO2025060553A1 - Use of maprotiline together with ctla4 antibody in preparation of anti-tumor drug - Google Patents

Abstract

A use of maprotiline, or a pharmaceutically acceptable salt thereof, together with a CTLA4 antibody in the preparation of an anti-tumor drug, the tumor being selected from colorectal cancers. It has been found that MAP, by means of targeting mTOR, can promote the ubiquitination of PD-L1 in colorectal cancer cells, causing the PD-L1 to degrade, thereby activating the immune system to exert an anti-tumor effect.

Description

Application of maprotiline combined with CTLA4 antibody in the preparation of anti-tumor drugs Technical Field

The present invention belongs to the field of medical technology, and in particular, relates to an application of maprotiline in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

Background Art

Maprotiline hydrochloride (MAP) is a tetracyclic antidepressant drug approved by the FDA. It can relieve mental retardation and produce antidepressant effects by blocking the reuptake of norepinephrine by the central presynaptic membrane. In addition, it also has anti-anxiety, sedative and visual protection effects. Currently, there are no reports on the use of MAP in cancer immunotherapy.

Chemical structure of Maprotiline hydrochloride

Currently, a variety of monoclonal antibodies targeting CTLA4, PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) and the China National Medical Products Administration (NMPA) for the clinical treatment of various tumors. These monoclonal antibodies have brought lasting and effective clinical benefits to many patients and changed the treatment pattern of various tumors. However, the vast majority of tumor patients still cannot benefit from them. There are still many limitations in current monoclonal antibody therapy, such as the lack of sensitive predictive markers for clinical application, the low overall response rate of patients to antibody therapy, and the existing efficacy evaluation indicators are not suitable for objective evaluation of tumor immunotherapy and strong toxic side effects. Small molecule inhibitors are a potential alternative because they can not only penetrate the cell membrane more easily and directly intervene in the PD-1/PD-L1 signaling pathway, but also have many advantages such as non-immunogenicity, low price, oral administration, and easy storage and transportation, which may help overcome certain treatment limitations and provide a wider range of treatment options. Therefore, the screening and development of small molecule inhibitors still have important potential. Although newly developed small molecule compounds are more targeted, the synthesis process is more complicated and their clinical safety also needs further verification. Repurposing approved drugs can reduce costs and significantly shorten the research cycle. Since drug toxicology, pharmacokinetic studies and clinical trials have been completed, these drugs with confirmed efficacy can benefit patients more quickly. In addition, in order to improve the response of cancer patients to PD-L1/PD-1 inhibitors, some clinical trials are studying PD-L1/PD-1 inhibitors in combination with chemotherapy, targeted therapy and The combination of other immunotherapies can improve the therapeutic effect. Therefore, it is a new idea for immunotherapy to find drugs with PD-L1 inhibitory effects from the old drugs on the market and combine them with CTLA4 antibodies.

PD-L1 (Programmed Death-Ligand 1), also known as CD274, is a protein that plays an important role in the immune system. PD-L1 is normally found on the surface of many different types of cells, both immune and non-immune. Its main function is to regulate the activity of the immune system by interacting with PD-1 (Programmed Death Receptor), which helps maintain the balance of the immune system. In some cancers, tumor cells overexpress PD-L1. This is a strategy to escape the attack of the immune system because when PD-L1 binds to PD-1, the immune system's ability to attack tumor cells is weakened. This phenomenon is called "immune escape" or "immunosuppression." In order to overcome the inhibitory effect of PD-L1 in tumor cells, anti-PD-L1 antibodies, such as pembrolizumab and nivolumab, have been developed. These drugs can bind to PD-L1 and prevent the interaction between PD-L1 and PD-1, thereby releasing the inhibition of T cells and enhancing the immune system's ability to attack cancer cells. This treatment method is called immune checkpoint inhibitor therapy. Although PD-L1 antibody therapy can effectively enhance the activity of the immune system, it may also cause some immune-related side effects, such as immune thyroiditis, skin reactions, gastrointestinal problems, etc. Therefore, there are certain risks in receiving PD-L1 antibody treatment.

In addition, the expression level of PD-L1 is regulated by many factors, including gene level, immune regulation, microenvironment, drug treatment, protein degradation pathway and epigenetics, such as gene mutation, immune system activity and regulatory factors, cells and signaling molecules in the tumor microenvironment, PD-L1 ubiquitination, DNA methylation and histone modification, which will directly affect the expression level of PD-L1. Understanding these factors will help to better understand the role of PD-L1 in immune regulation and tumor immune escape, and is expected to provide useful information for individualized tumor treatment and the development of new treatment strategies.

Clinically, antibody combined immunotherapy has become an important cancer treatment strategy that can enhance the immune system's attack on cancer cells. This combination therapy usually involves the use of antibodies with different mechanisms to enhance the activity of the immune system and improve the treatment of cancer. The following are some common antibody combined immunotherapy methods and effects: anti-PD-1/PD-L1 combined with anti-CTLA4 therapy, anti-PD-1/PD-L1 antibodies can relieve the PD-1/PD-L1 inhibitory effect between T cells and cancer cells, while CTLA4 antibodies can enhance the activity of T cells. This combination therapy has achieved remarkable success in many cancer types such as melanoma; anti-PD-1/PD-L1 combined with targeted drugs, sometimes, anti-PD-1/PD-L1 antibodies can also be used in combination with targeted drugs to enhance the therapeutic effect. For example, for patients with non-small cell lung cancer with EGFR mutations, the combined use of EGFR inhibitors and anti-PD-1/PD-L1 antibodies may produce better therapeutic effects; anti-PD-1/PD-L1 combined with anti-tumor antigen (TAA) vaccines, the combination treatment strategy of anti-PD-1/PD-L1 antibodies and anti-tumor antigen vaccines aims to stimulate the patient's own immune system to produce a specific response to cancer cells. This combination treatment may enhance the vaccine-induced immune response and improve the immune killing effect on cancer. In addition to the above methods, there are also a variety of other combination strategies of immunotherapy drugs and antibodies, such as anti-LAG-3 antibodies, anti-TIM-3 antibodies, etc., which are also under study. These combination treatments are designed to target different immune regulatory pathways, enhancing the diversity of treatment methods. However, the effect of antibody combined immunotherapy is generally Treatment options often vary depending on cancer type, patient characteristics, and treatment history; therefore, treatment choices often need to be tailored to the patient's specific circumstances and the latest research findings on immunotherapy.

The patent application with publication number CN104546813A discloses the use of maprotiline hydrochloride in the preparation of drugs for inhibiting the metastasis and spread of tumor cells. The drugs prepared by maprotiline hydrochloride inhibit the metastatic ability of tumor cells. Maprotiline hydrochloride is a low-toxic drug that can play a greater blocking role in the process of tumor spread. At the same time, other related tumor drugs are used for treatment, which can greatly reduce the death of normal cells; while the existing tumor drugs are mostly cytotoxic drugs, a large number of normal cells die together with the tumor cells.

Summary of the invention

The purpose of the present invention is to provide an application of maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

The first aspect of the present invention provides a use of maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

The tumor is selected from colorectal cancer.

The pharmaceutically acceptable salt is an acid addition salt formed by maprotiline and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

The second aspect of the present invention provides a use of maprotiline or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in combination with a CTLA4 antibody in the preparation of an anti-tumor drug.

The tumor is selected from colorectal cancer.

In the present invention, maprotiline or a pharmaceutically acceptable salt thereof is used as a PD-L1 inhibitor to target mTOR to promote the ubiquitination degradation of PD-L1 in cancer cells, activate T cell immunity, and exert an anti-cancer effect. There is no related report at present.

Under normal conditions, the main function of the PD-1/PD-L1 inhibitory co-stimulatory signal is to prevent T cells from uncontrolled overactivation and attacking normal cells. The high level of PD-L1 expressed on the surface of tumor cells inhibits T cell function and promotes T cell exhaustion by binding to PD-1 on the surface of T cells, allowing tumor cells to escape T cell immune attack. Maprotiline or its pharmaceutical salt in the present invention is used as a PD-L1 inhibitor to prevent tumor cell immune escape, and its mechanism of action is consistent with that of PD-1 antibody and PD-L1 antibody, but CTLA4 terminates the immune response by binding to receptors on the surface of antigen cells. CTLA4 antibodies cause T cells to proliferate and attack tumor cells in large quantities by inhibiting CTLA-4 molecules. Therefore, CTLA4 antibodies are selected for combined use with maprotiline or its pharmaceutical salt.

The pharmaceutically acceptable salt is an acid addition salt formed by maprotiline and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, Phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

The third aspect of the present invention provides a pharmaceutical preparation, which is prepared by combining maprotiline or a pharmaceutically acceptable salt thereof with a CTLA4 antibody.

Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

The use of maprotiline or its pharmaceutically acceptable salt in the preparation of anti-tumor drugs provided by the present invention verifies for the first time through in vivo and in vitro experiments that it exerts anti-tumor activity through immunomodulatory function. It verifies at the cellular level that MAP reduces the expression level of PD-L1; verifies at the cellular level the in vitro killing ability of T cells; and verifies at the animal level the immunotherapy ability of MAP.

The use of maprotiline or a pharmaceutically acceptable salt thereof provided by the present invention in the preparation of anti-tumor drugs proves that MAP has an immunotherapeutic effect and that the dose producing the anti-tumor effect is safe; it proves that MAP promotes the killing of cancer cells by T cells by downregulating PD-L1 in vivo and in vitro; the combination of MAP and CTLA4 antibody can produce a synergistic anti-cancer effect, and the anti-tumor effect is significantly enhanced. This "old drug new use" model provides a new alternative for the clinical treatment of colorectal cancer.

The invention provides an application of maprotiline or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug. The breakthrough of the invention is that it is the first discovery that MAP can promote the ubiquitination of PD-L1 in colorectal cancer cells to degrade it by targeting mTOR, thereby activating the immune system to exert an anti-tumor effect.

In order to study whether the combination of MAP and CTLA4 antibodies has a synergistic anti-tumor effect. The present invention subcutaneously inoculated MC38 in C57BL/6J (female) mice, and treated the mice using corn oil, MAP (40mg/kg), anti-PD-1 (100ug/mouse), anti-CTLA4 (100ug/mouse) and MAP combined with anti-CTLA4. The results showed that compared with the use of MAP alone or anti-CTLA4 alone, the growth rate and volume of the tumor were further improved after the combined treatment of MAP and anti-CTLA4. In addition, the flow cytometric analysis results of tumors in different groups of mice showed that the MAP combined with anti-CTLA4 group had the least MDSCs (CD11b ⁺ Gr-1 ⁺ ) and Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell populations and the highest granzyme B levels. Therefore, the results of the study showed that MAP transformed the immune microenvironment from immunosuppression to immune activation, and MAP and anti-CTLA4 synergistically inhibited the proliferation of colorectal cancer cells and promoted anti-tumor effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the effect of detecting cytotoxicity of RKO and MC38 cells using a CCK-8 kit.

FIG. 2 is a schematic diagram of a BSA protein standard curve.

FIG3 is a schematic diagram showing that MAP can downregulate PD-L1 levels in RKO and MC38 cells.

FIG. 4 is a schematic diagram of detecting the expression of PD-L1 on the membrane of colon cancer cells by flow cytometry.

FIG5 is a schematic diagram showing the results of immunofluorescence detection of the down-regulation effect of MAP on PD-L1 on the cancer cell membrane.

FIG6 is a schematic diagram showing the results of measuring the in vitro killing effect of MAP on T cells by crystal violet staining.

FIG. 7 is a schematic diagram showing the anti-tumor effects of different doses of MAP on C57BL/6J mice.

FIG8 is a schematic diagram showing the anti-tumor effects of the control group and MAP (40 mg/kg) on nude mice.

FIG9 is a schematic diagram showing the anti-tumor effect of MAP in combination with CTLA4 antibodies.

FIG. 10 is a schematic diagram of the immunohistochemical analysis results showing that MAP can be combined with CTLA4 antibody to produce a synergistic anti-tumor effect.

DETAILED DESCRIPTION

In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

Example 1

Study on the effect of MAP on the vitality of colorectal cancer cells in vitro

1.1 Experimental Materials:

Colorectal cell lines and their culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-MEM (MeilunBio) and DMEM (MeilunBio) culture media containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO _2. The compound Maprotiline hydrochloride (Cat. No.: HY-B0444) was purchased from MedChemExpress (MCE); other reagents: CCK-8 kit (C0048L) and DMSO (ST1276) were all products of Beyotime.

1.2 Experimental methods

The cytotoxicity of MAP to the above tumor cell lines was detected by CCK-8. The specific steps are as follows:

1. Sample preparation: Dissolve MAP in dimethyl sulfoxide to obtain a solution with a concentration of 10 μM;

2. After trypsin digestion and washing, the cells were suspended in MEM or DMEM medium containing 10% fetal bovine serum. The number of live cells was counted by trypan blue staining exclusion method, and the density of the cell suspension was adjusted to 1×10 ⁵ cells/mL;

3. Add 100 μl of cells to each well of a flat-bottom 96-well plate, with a total number of cells in each well of 8×10 ³ . Incubate in a 37°C, 5% CO ₂ cell culture incubator until the cells are completely attached;

4. Change the medium and add drugs. Add 100 μl of sample diluted with cell growth medium to each well, so that the final concentrations of the reaction are 1, 5, 10 and 20 μM respectively. Add the corresponding amount of DMSO to the cells without drug addition as a control, and the wells containing cells without drug and DMSO as blanks. Perform three parallel tests for each point;

5. Incubate the treated cells in a 37°C, 5% CO ₂ cell culture incubator for 24 hours;

6. Replace the culture medium in each well with serum-free culture medium and add 10 μL CCK-8 reagent, continue to incubate in the incubator for 2-5 hours, and measure the absorbance (OD value) at a wavelength of 450 nm with an enzyme reader;

7. Viability calculation: Cell viability (%) = [A1-A3]/[A2-A3] × 100, A1: OD value of wells with cells, CCK-8 solution and drug solution, A2: OD value of wells with cells, CCK-8 solution but no drug solution, A3: OD value of wells without cells;

8. GraphPad Prism 9.0.1 software was used to perform statistical analysis and plot the data to obtain the IC ₅₀ .

1.3 Experimental Results

Since the CCK-8 reagent contains WST-8, it is reduced to a highly water-soluble yellow formazan dye by dehydrogenases in the cell mitochondria under the action of an electron carrier (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells, so this property can be used to directly analyze cell proliferation and toxicity. If the drug has an inhibitory effect on the cells, the color will be lighter. The results are shown in Figure 1, which is a diagram of the effect of using the CCK-8 kit to detect cytotoxicity in RKO and MC38 cells. The cell survival rate of the control group incubated with CCK-8 reagent (10μL) for 4 hours was the highest, and MAP had little effect on cell proliferation after 24 hours of treatment of colorectal cancer cells at 1μM, 5μM, and 10μM, indicating that 10μM is a safe concentration of MAP. As can be seen from the figure, the IC ₅₀ of MAP on colorectal cancer cells RKO and MC38 are 17.71μM and 22.94μM, respectively. This shows that MAP has no toxic effect on RKO and MC38 cells at 10μM.

The present invention will take colon cancer cells (RKO and MC38) as an example to further illustrate that MAP exerts anti-tumor effects by reducing the expression of PD-L1 in tumor cells.

Example 2

Study on downregulation of PD-L1 expression in colon cancer cells by MAP

2.1 Experimental Materials:

Colorectal cell lines and their culture: Human colorectal cancer cells RKO and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-MEM (MeilunBio) and DMEM (MeilunBio) culture medium containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO _2. PD-L1 antibody was purchased from Abcam, catalog number ab203103; BCA kit was from Biotech (P0009).

2.2 Experimental methods

2.2.1 Protein immunoblotting

The cells with good growth status were seeded in a 6-well plate at a density of 4×10 ⁵ cells/well, and the suspension volume in each well was 2 mL. The six-well plate with cells was placed in a 37°C incubator for culture. After the cells were cultured until they were completely attached to the wall, MAP was administered at 5, 10, and 20 μM. After continuing to culture for 24 hours, the cells were collected for experiments.

The collected cells were rinsed twice with cold PBS, PBS was aspirated, and the corresponding amount of lysis buffer with PMSF was added according to different culture plates and actual cell numbers. After incubation on ice for 15 minutes, centrifuge at 12000 rpm for 15 minutes in a 4°C centrifuge. The supernatant was gently aspirated and transferred to a newly pre-cooled centrifuge tube and placed on ice. This is the protein sample, and the precipitate was discarded. The BCA method is used for protein quantification. Protein denaturation generally uses 5×SDS gel reduction loading buffer (5×Loading Buffer), which is mixed with the sample at a ratio of 1:4 and incubated in a metal bath at 95°C for 10 minutes. After cooling, the sample is stored at -20°C.

The BCA assay method is as follows (Biyuntian BCA kit):

According to the number of samples, prepare the working solution of reagent A and reagent B in the kit at a ratio of 50:1, mix thoroughly and add 200μL BCA working solution to each well of a flat-bottom 96-well plate, and avoid light. Prepare the standard in the kit into a 25mg/mL protein standard solution, and draw a standard curve according to the concentration in the table below, with BSA concentration (mg/mL) as the horizontal axis and absorbance as the vertical axis.

Table 1 Standard preparation of koji protein

The standard curve is shown in FIG2 , which is a schematic diagram of the BSA protein standard curve.

When quantifying the protein of the sample, take 2 μL of each sample and add it to 198 μL of BCA working solution, then shake it in a 37°C incubator for 30 minutes, and finally detect the absorbance of the sample on an enzyme reader, and substitute the value into the protein standard curve to calculate the protein concentration of the sample.

Prepare the protein sample according to the molecular weight of the target protein, prepare the corresponding protein gel for electrophoresis. During electrophoresis, first use 80V constant voltage electrophoresis for about 30min. When the indicator bromophenol blue enters the separation gel, use 120V constant voltage electrophoresis. When the indicator reaches about 0.5cm from the lower end of the gel, turn off the power, take out the gel plate, and transfer the mold with methanol-activated PVDF membrane. In an ice bath, 250mA constant current for 1.5h. After the transfer, quickly take out the PVDF membrane, place it in a 5% skim milk solution for 1h, take out the membrane, and wash it three times with TBST solution on a shaker, 5min each time. Then incubate it with the corresponding primary antibody (PD-L1) at 4℃ overnight. After the primary antibody incubation is completed, wash the membrane with TBST 3 times, 5min each time, and then incubate the secondary antibody, incubate at 4℃ for 2h or shake and incubate at room temperature (22-25℃) for 1h. Finally, wash the membrane with TBST 3 times and then develop it in the developer.

2.3 Experimental Results

RKO and MC38 cells were treated with 5μM, 10μM and 20μM MAP for 24 hours. The results are shown in Figure 3, which is a schematic diagram of MAP downregulating PD-L1 levels in RKO and MC38 cells. In the figure, A indicates that after RKO cells were treated with 5μM, 10μM and 20μM MAP for 24 hours, the PD-L1 protein level in RKO cells decreased with the increase of MAP administration concentration, and B indicates that after MC38 cells were treated with 5μM, 10μM and 20μM MAP for 24 hours, The PD-L1 protein level in MC38 cells decreased with the increase of MAP administration concentration. C indicates that 10 μM MAP was used to treat RKO cells for 3 hours, 6 hours, 9 hours, 12 hours and 24 hours respectively. The results showed that MAP could time-dependently reduce the expression of PD-L1 in RKO cells. D indicates that 10 μM MAP was used to treat MC38 cells for 3 hours, 6 hours, 9 hours, 12 hours and 24 hours respectively. The results showed that MAP could time-dependently reduce the expression of PD-L1 in MC38 cells.

As can be seen from A and B, the effects of 5μM, 10μM and 20μM MAP on reducing PD-L1 in colorectal cancer cells (RKO and MC38) increase successively, indicating that it is dose-dependent; as can be seen from C and D, the downregulation of PD-L1 in colorectal cancer cells (RKO and MC38) by 10μM MAP is time-dependent. The results showed that MAP significantly reduced the expression of PD-L1, and this effect was related to the concentration of MAP. 10μM MAP was used to treat RKO cells, and the treatment was performed for 3 hours, 6 hours, 9 hours, 12 hours and 24 hours respectively. The results showed that MAP was able to reduce the expression of PD-L1 in cells in a time-dependent manner.

Example 3

Flow cytometry was used to detect the downregulation of MAP on PD-L1 on cancer cell membrane

3.1 Experimental Materials:

Colorectal cell lines and their culture: Human colorectal cancer cells RKO, DLD1, HT29 and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in MEM, RPMI1640, McCoy's5A and DMEM (MeilunBio) medium containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO _2. The flow cytometry antibodies used were purchased from Biolegend, including mouse PD-L1 antibody and human PD-L1 antibody, with catalog numbers 124307 and 329706, respectively.

3.2 Experimental methods

3.2.1 Flow cytometry detection of tumor cell membranes shows PD-L1 abundance

Colorectal cancer cells in good condition were inoculated in a 12-well plate at a density of 2.5×10 ⁵ cells per well, with 1 mL of cell suspension per well. After the cells were completely attached to the wall, MAP was added for 24 hours, and then the culture medium was removed. The cells digested with trypsin were collected in an EP tube, centrifuged at 1200 rpm and 4°C for 3 minutes in a centrifuge, the supernatant was removed, and the cells were washed twice with pre-cooled PBS, and then centrifuged at 1200 rpm for 3 minutes at 4°C. The supernatant was removed, and 100 μL of PE anti-human CD274 antibody or PE anti-mouse CD274 antibody diluted with PBS was added to each tube and incubated at 4°C for 30 minutes. After the incubation, 400 μL of PBS was added for washing, and the cells were centrifuged at 1200 rpm and 4°C for 3 minutes. The supernatant was removed, and 500 μL of PBS was added to resuspend the cells and transferred to a flow tube, and then detected in a flow cytometer.

3.3 Experimental Results

PD-L1 membrane expression plays an important role in immune regulation, especially in regulating T cell activity and the balance of the immune system. Cancer cells mainly escape immunity by binding PD-L1 on their membranes to PD-1 on the surface of T cells. Therefore, only by reducing the PD-L1 protein on the cancer cell membrane can the anti-cancer effect be exerted through immunity.

The results are shown in FIG4 , which is a schematic diagram of flow cytometry detection of PD-L1 expression on the membrane of colon cancer cells. In the figure, A indicates that after RKO cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of RKO cells decreased as the MAP administration concentration increased; B indicates that after DLD1 cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of DLD1 cells decreased as the MAP administration concentration increased; C indicates that after HT29 cells were first treated with 50ng/mL IFN-γ for 2 hours and then treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of HT29 cells decreased as the MAP administration concentration increased; D indicates that after MC38 cells were treated with 5μM, 10μM, 15μM and 20μM MAP for 24 hours, the abundance of PD-L1 on the membrane surface of MC38 cells decreased as the MAP administration concentration increased.

As can be seen from Figures A-D, MAP reduces the expression of PD-L1 on the cell membrane of colon cancer cells (RKO, DLD1, HT29 and MC38) in a concentration-dependent manner (5μM, 10μM, 15μM and 20μM). In this experiment, by flow cytometry, MAP can reduce the abundance of PD-L1 on the cell membrane of colorectal cancer cells RKO, DLD1, HT29 and MC38 in a concentration-dependent manner, blocking the binding of PD-L1 to PD-1, and achieving the effect of immunotherapy.

Example 4

Immunofluorescence detection of MAP downregulation of PD-L1 on cancer cell membrane

4.1 Experimental Materials:

Colorectal cell lines and their culture: Human colorectal cancer cells RKO, DLD1 and mouse colorectal cancer cells MC38 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in MEM, RPMI1640 and DMEM medium (MeilunBio) containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO _2. Immunofluorescence anti-PD-L1 antibody was purchased from Proteintech (Cat. No. 66248-1-Ig).

4.2 Experimental methods

1. Cell culture and washing: Cancer cells in good condition were seeded in a 12-well plate at a density of 2.5×10 ⁵ cells per well, 1 mL per well, and placed in cell culture medium until the cells were completely attached to the wall. MAP was added for 24 h, the supernatant was removed, and the cells were washed 3 times with PBS;

2. Fixation and blocking: Fix the cells in the culture plate with 4% paraformaldehyde solution for 15 minutes, remove the fixative, wash with PBS three times, 5 minutes each time, add 5% BSA to the sample wells of the 12-well plate for blocking for 1 hour, and wash twice with PBS;

3. Primary antibody incubation: Add diluted PD-L1 primary antibody, incubate overnight at 4°C, and wash 3 times with TBST, 5 minutes each time;

4. Secondary antibody incubation: Finally, add red fluorescent antibody and incubate at room temperature in the dark for 1 hour. Add DAPI for staining for 10 minutes before taking pictures. Wash twice with TBST buffer, 5 minutes each time.

5. Analyze the results after laser confocal imaging.

4.3 Experimental Results

The intensity of red in immunofluorescence represents the expression of PD-L1 on the cell membrane. The stronger the fluorescence, the higher the expression of PD-L1, and the weaker the fluorescence, the weaker the expression of PD-L1. The results are shown in Figure 5, which is a schematic diagram of the results of immunofluorescence detection of MAP's downregulation of PD-L1 on the cancer cell membrane. After MAP treatment of colorectal cancer cells, the red color of immunofluorescence staining can show the expression of PD-L1. The higher the concentration of MAP, the less red the immunofluorescence staining, indicating that MAP reduces the abundance of colorectal cancer cells (PD-L1 on the membrane of RKO, DLD1 and MC38) in a concentration-dependent manner. The immunofluorescence results showed that the red fluorescence of colorectal cancer cells treated with MAP was reduced in a concentration-dependent manner (5μM, 10μM, 15μM and 20μM), which again showed that MAP can reduce the expression of PD-L1 on the cell membrane of colon cancer cells (RKO, DLD1 and MC38) and prevent the transport of PD-L1 to the plasma membrane.

Example 5

Study on the effect of MAP on enhancing the killing effect of T cells on colon cancer cell line RKO in vitro

5.1 Experimental Materials:

Human colorectal cell lines and their culture: Colorectal cancer cells RKO were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in MEM medium (purchased from MeilunBio) containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% _CO2 ; NK-92 cells were purchased from ATCC and cultured in a special NK cell culture medium (Shanghai Xuhe Biology, catalog number: s-22). Crystal violet solution was from Biyuntian (catalog number: C0121).

5.2 Experimental methods

The in vitro killing effect of MAP on RKO cell line was measured by NK-92 method. The specific steps are as follows:

1. Cell culture and washing: Cancer cells in good condition were inoculated into a 12-well plate at a density of 3×10 ⁵ cells/mL, 1 mL per well, and placed in cell culture until the cells were completely attached to the wall, and then treated with MAP for 24 hours;

2. Add NK-92 cells at a ratio of 1:0.5 and culture for an appropriate amount of time. During this period, pay attention to the killing situation to avoid excessive killing;

3. Discard the supernatant, wash with PBS three times, fix the cells with 4% paraformaldehyde, wash with PBS three times, add crystal violet solution to stain for 25 minutes, then wash away the excess crystal violet with PBS, turn the 12-well plate upside down, and wait for it to dry naturally;

4. Use Cytation5 to collect images and acquire data, and use ImageJ software to perform statistical analysis on the remaining tumor cells.

5.3 Experimental Results

In order to further evaluate the in vitro anti-tumor effect of MAP, RKO and NK92 cells were co-cultured, and the surviving tumor cells were detected by crystal violet staining. The results are shown in Figure 6, which is a schematic diagram of the results of crystal violet staining to determine the in vitro killing effect of MAP on T cells. A represents the treatment of RKO cells with 5μM and 10μM MAP for 24 hours. After 5 hours, NK-92 cells were added, and MAP could increase the killing ability of NK-92 cells against tumor cells in a concentration-dependent manner. The results in A show that MAP also enhances the killing effect of NK92 cells on colorectal cancer cells RKO in a concentration-dependent manner; B is a statistical schematic diagram of apoptotic cells in Figure A. It can be seen from Figures A and B that MAP can increase the killing effect of NK-92 cells on tumor cells in a concentration-dependent manner. The results show that at a safe and effective concentration, MAP can significantly enhance the killing effect of T cells on tumor cells and reduce the survival rate of tumor cells. According to Figure 6, it is not difficult to conclude that MAP enhances the cytotoxicity of T cells to cancer cells and induces cell apoptosis by downregulating the expression of PD-L1 in colon cancer cells.

Example 6

Antitumor effect of MAP in vivo

6.1 Experimental Materials:

Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured and passaged in DMEM (MeilunBio) medium containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO ₂ .

Animals: C57BL/6J mice (female), purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., certificate number (20220009006037).

Other reagents: Maprotiline hydrochloride (catalog number: HY-B0444) was purchased from MedChemExpress (MCE); DMSO (batch number: 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd.).

Samples: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

6.2 Experimental methods

Colon cancer cell MC38 was inoculated into 6-8 week old female C57BL/6J mice (8×10 ⁵ cells/mouse) to establish a subcutaneous tumor model. After the tumor volume reached 50 mm ³ , the mice were divided into 4 groups: (1) control group; (2) low MAP (10 mg/kg) group; (3) medium MAP (20 mg/kg) group; (4) high MAP (40 mg/kg) group. The mice were given drugs by oral gavage every day for 16 days, and the tumor volume and body weight of the mice were measured every other day. The formula for calculating tumor volume is: tumor volume (mm ³ ) = (length) × (width) ² × 1/2.

6.3 Experimental Results

MAP treatment showed a significant inhibitory effect on the growth of mice inoculated with MC38 tumors, and was accompanied by concentration dependence. The best anti-tumor effect was achieved when the MAP administration concentration was 40 mg/kg. The results are shown in Figure 7, which is a schematic diagram of the anti-tumor effect of different doses of MAP on C57BL/6J mice, among which 40 mg/kg had the best anti-tumor effect. Among them, A is a comparison of the actual tumors of mice after treatment in different groups, and the results show that the anti-tumor effect of MAP is concentration-dependent; B is a schematic diagram of the tumor volume curve of different groups, and the results show that MAP inhibits tumor growth in a concentration-dependent manner; C is the curve of each group The other is a comparison chart of tumor weight, the results show that with the increase of MAP administration concentration, the tumor weight is smaller; D is a schematic diagram of the weight change of mice in different groups, the results show that MAP has no significant effect on the weight of mice; E is a flow cytometry analysis diagram of Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell populations in tumor tissues of different groups; F is a statistical diagram of Foxp3 in different groups, E and F show that with the increase of MAP administration dose, the proportion of Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell population in tumor cells decreases successively; G is a flow cytometry analysis diagram of MDSCs (CD11b ⁺ Gr-1 ⁺ ) cell population activated in TILs in tumors of different groups; H is a statistical diagram of Gr-1 in different groups, G and H show that with the increase of MAP administration dose, the proportion of MDSCs (CD11b ⁺ Gr-1 ⁺ ) The proportion of the cell population decreases successively; I is a flow cytometry analysis diagram of granzyme B in tumors of different groups; J is a statistical diagram of granzyme B in different groups, and I and J show that with the increase of MAP administration dose, the proportion of granzyme B in tumor cells increases successively.

As shown in Figure 7A, as the dosage of MAP increases, its inhibitory effect on mouse tumors gradually increases. The inhibition rates of MAP at dosages of 10, 20 and 40 mg/kg were 22.19%, 53.11% and 70.16%, respectively (Figure 7B). The anti-tumor efficacy of MAP was further verified by comparing the tumor weights between different groups (Figure 7C). Taking MAP had almost no effect on the weight of mice (Figure 7D), indicating that MAP had no systemic toxicity to mice. In addition, in the tumor microenvironment, myeloid-derived suppressor cells (MDSCs) and Tregs promote tumor immune escape by strongly inhibiting T lymphocyte immunity. Activated MDSCs and Tregs can express a large amount of PD-L1 to interact with PD-1 on T cells, and ultimately lead to their exhaustion. By flow cytometry analysis of tumor tissues of mice in different groups, it was found that the level of granzyme B, an indicator of cytotoxic T cell activation, increased with the increase of MAP dosage, indicating that MAP can stimulate the activity of cytotoxic T lymphocytes (Figure 7I). Moreover, compared with the tumors in the control group, the populations of activated MDSCs (CD11b ⁺ Gr-1 ⁺ ) and Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) in TILs in MAP-treated tumors were significantly reduced with the increase of MAP (as shown in Figure 7 EH). These results indicate that MAP can effectively activate tumor-infiltrating T cells to exert anti-tumor effects.

Example 7

Antitumor effect of MAP in nude mice

7.1 Experimental Materials:

Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured and passaged in DMEM (MeilunBio) medium containing 10% fetal bovine serum (Biological Industrie) at 37°C and 5% CO ₂ .

Animals: Nude mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., certificate number (20220009007529).

Other reagents: Maprotiline hydrochloride (catalog number: HY-B0444) was purchased from MedChemExpress (MCE); DMSO (batch number: 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd. company).

Samples: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

7.2 Experimental methods

Colon cancer cells MC38 were inoculated into the armpits of 6-8 week old female nude mice (8×10 ⁵ cells/mouse) to establish a subcutaneous tumor model. When the tumor volume reached about 50 mm ³ , the mice were randomly divided into a control group and a treatment group with MAP (40 mg/kg). The mice were given drugs by oral gavage every day for 12 days, and the tumor volume and body weight of the mice were measured every other day. The formula for calculating the tumor volume is: tumor volume (mm ³ ) = (length) × (width) ² × 1/2.

7.3 Experimental Results

To further confirm that MAP exerts its anti-tumor effect through immune pathways, this experiment used immunodeficient mouse experiments to verify whether MAP has anti-tumor effects. The results are shown in Figure 8, which is a schematic diagram of the anti-tumor effects of the control group and MAP (40 mg/kg) on nude mice. Among them, A is a comparison of the actual sizes of tumors in the control group and the drug-treated group, indicating that MAP has no anti-tumor effect in immune-deficient nude mice; B is a schematic diagram of the tumor growth curves of the two groups of mice, indicating that MAP has no effect on tumor growth in nude mice; C is a statistical diagram of the tumor weight of the two groups of mice, indicating that MAP does not affect the weight of nude mouse tumors; E is a schematic diagram of HE staining of the organs of the two groups of mice, indicating that MAP has no effect on the morphology of the heart, liver, spleen, lung, and kidney of mice.

As shown in Figure 8, in nude mice lacking T cells, MAP lost its inhibitory effect on MC38 tumors, indicating that the anti-tumor effect of MAP is mainly produced by activating the immune system in the body and increasing T cell infiltration. In addition, by comparing the results of HE staining of the internal organs of mice in different groups, it can be determined that MAP has no toxic effect on mice at this dose.

Example 8

MAP can combine with CTLA4 antibody to produce synergistic anti-tumor effects

8.1 Experimental Materials:

Tumor line: Mouse colorectal cancer cells (MC38) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured and passaged in DMEM (MeilunBio) medium containing 10% fetal bovine serum (Biological Industries) at 37°C and 5% CO ₂ .

Animals: C57BL/6J mice (female), purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., certificate number (20220009009804).

Other reagents: Maprotiline hydrochloride (catalog number: HY-B0444) was purchased from MedChemExpress (MCE); anti-mouse-PD-1 antibody (Invivogen, catalog number: BE0146); anti-mouse-CTLA4 (Invivogen, catalog number: BP0032); DMSO (batch number: 20030415) was a product of Shanghai Chemical Reagent Company; corn oil (Golden Sun Grain and Oil Co., Ltd.).

Samples: MAP was dissolved in DMSO and corn oil at a 10% corn oil dose.

8.2 Experimental methods

In order to study whether the combination of MAP and anti-CTLA4 (CTLA4 antibody) has a synergistic anti-tumor effect, colon cancer cell MC38 was inoculated into 6-8 week old female C57BL/6J mice (8×10 ⁵ cells/mouse) to establish a subcutaneous tumor model. When the tumor volume reached 50 mm ³ , the mice were divided into five groups: (1) control group; (2) anti-PD-1 group; (3) anti-CTLA4 group; (4) MAP (40 mg/kg) group; (5) MAP (40 mg/kg) + anti-CTLA4 group (100 μg/mouse). The administration days were 16 days. MAP was administered by gavage, and the antibody was injected intraperitoneally every five days. A total of three injections of antibody were given, each time with 100 μg of intraperitoneal injection. The tumor volume and body weight of the mice were measured every other day. The tumor volume calculation formula is: tumor volume (mm ³ ) = (length) × (width) ² × 1/2.

8.3 Experimental Results

Since the adverse reactions after the combination of PD-1 antibody and CTLA4 antibody are large, the combination of small molecule drugs and CTLA4 antibodies has become a cancer immunotherapy strategy aimed at enhancing the immune system's attack on tumors. The goal of this combination therapy is to improve the treatment effect by simultaneously intervening in multiple immune regulatory pathways.

Figure 9 is a schematic diagram of the anti-tumor effect of MAP combined with CTLA4 antibodies. Among them, A is a schematic diagram of the actual tumor size of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has the best anti-tumor effect; B is a schematic diagram of the tumor volume curve of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has the most obvious inhibitory effect on tumor growth; C is a schematic diagram of the comparison of tumor weights in each group, and the results show that the tumor weight of the combination of MAP and CTLA4 antibodies is the lightest; D is a schematic diagram of the weight change of mice in different groups, and the results show that the combination of MAP and CTLA4 antibodies has a certain effect on the weight of mice; E is a flow cytometry analysis of Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell populations in tumor tissues of different groups; F is a statistical diagram of Foxp3 in different groups; E and F show that the proportion of Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell populations in tumor tissues after the combination of MAP and CTLA4 antibodies is the smallest; G is the activated MDSCs (CD11b ⁺ Gr-1 ⁺ ) is a schematic diagram of flow cytometry analysis of cell populations; H is a statistical schematic diagram of Gr-1 in different groups; G and H indicate that the MDSCs (CD11b ⁺ Gr-1 ⁺ ) cell population accounts for the least in tumor tissues after the combination of MAP and CTLA4 antibodies; I is a schematic diagram of flow cytometry analysis of granzyme B in tumors of different groups; J is a statistical schematic diagram of granzyme B in different groups; I and J indicate that the MDSCs (CD11b ⁺ Gr-1 ⁺ ) cell population accounts for the highest proportion in tumor tissues after the combination of MAP and CTLA4 antibodies.

Figure 10 is a schematic diagram of the immunohistochemical analysis results showing that MAP can be combined with CTLA4 antibodies to produce a synergistic anti-tumor effect. Among them, A is a schematic diagram of the results of immunohistochemical analysis of tumor tissues in different groups, and Figure B is a schematic diagram of the quantification results of Figure A. The results of Figures A and B show that the levels of CD3 (T cell marker), CD4 (regulatory helper T cells), CD8 (cytotoxic T cells) and C-caspase-3 (cleaved caspase-3) in the tumors of the group using MAP in combination with CTLA4 antibodies were the highest, while the levels of Ki-67 (proliferation marker), PD-L1 and FOXP3 (an immunosuppressive molecule) were reduced, indicating that MAP induces significant tumor cell apoptosis in mice. This shows that the combination of MAP and CTLA4 antibodies It can greatly enhance the infiltration of T cells in tumor tissue, and its effect is stronger than the use of PD-1 antibody alone, MAP alone, or CTLA4 antibody alone.

The results showed that compared with MAP alone or CTLA4 antibody alone, the growth rate and volume of the tumor were further improved after combined treatment with MAP and CTLA4 antibody (as shown in AC in Figure 9). However, there was a certain effect on the weight of mice after combined treatment (as shown in D in Figure 9). In addition, the flow cytometry analysis results of tumors in different groups of mice showed that the MAP combined with CTLA4 antibody group had the fewest MDSCs (CD11b ⁺ Gr-1 ⁺ ) and Tregs (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) cell populations and the highest granzyme B levels (as shown in EJ in Figure 9). Therefore, the results of the study indicate that MAP transforms the immune microenvironment from immunosuppression to immune activation, and that MAP and CTLA4 antibodies synergistically inhibit the proliferation of colorectal cancer cells and promote anti-tumor effects.

Colorectal cancer is a highly prevalent malignant tumor in humans, which seriously endangers human health. At present, the clinical treatment of colorectal cancer is still mainly surgery, radiotherapy, chemotherapy, and targeted drugs, but the overall efficacy is limited. Therefore, it is urgent to explore safe and effective new treatment strategies. Blocking the combination of PD-1 and PD-L1 is an important tumor immunotherapy method, but the expression of PD-L1 in tumor cells may affect the clinical efficacy of PD-1/PD-L1 related immune checkpoint therapy. Therefore, finding small molecules that target and regulate PD-L1 expression is expected to become a new treatment method. The present invention found through protein immunoblotting, flow cytometry and immunofluorescence experiments that maprotiline hydrochloride (MAP) can not only reduce the total protein level of PD-L1 in cancer cells, but also reduce the expression of PD-L1 on the cancer cell membrane, thereby activating the immune system to exert an anti-tumor effect. In addition, it was found that the anti-tumor effect of maprotiline hydrochloride combined with CTLA4 antibody was significantly enhanced, which provides a new alternative for the clinical treatment of colorectal cancer.

Advantages of the present invention: Small molecule drugs have many advantages, such as non-immunogenicity, low price, oral administration, easy storage and transportation, etc. These advantages may help to overcome the challenges faced by immune checkpoint inhibitors in some cases, including immune-related side effects, high treatment costs and the need for intravenous injection. Therefore, the screening and development of small molecule inhibitors still have important potential. In addition, compared with newly developed small molecule compounds whose synthesis process is more complicated and whose clinical safety also needs further verification, the reuse of approved and marketed drugs can reduce costs and significantly shorten the research cycle. Since the drug toxicology, pharmacokinetic studies and clinical trials of marketed drugs have been completed, these drugs with confirmed efficacy can benefit patients more quickly. Therefore, this "new use of old drugs" model has great prospects in future tumor immunotherapy.

The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims (4)

A use of maprotiline or a pharmaceutically acceptable salt thereof in combination with a CTLA4 antibody in the preparation of an anti-tumor drug, characterized in that the tumor is selected from colorectal cancer. The use of maprotiline or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug according to claim 1, characterized in that the pharmaceutically acceptable salt is an acid addition salt formed by maprotiline and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid. Disclosed is the use of maprotiline or a pharmaceutically acceptable salt thereof as a PD-L1 inhibitor in combination with a CTLA4 antibody in the preparation of an anti-tumor drug. A pharmaceutical preparation, characterized in that it is prepared by combining maprotiline or a pharmaceutically acceptable salt thereof with a CTLA4 antibody.